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Hindawi Publishing CorporationJournal of PathogensVolume 2012,
Article ID 781652, 6 pagesdoi:10.1155/2012/781652
Research Article
Rapid Detection and Identification of Yersinia pestis from
FoodUsing Immunomagnetic Separation and Pyrosequencing
Kingsley K. Amoako,1 Michael J. Shields,1 Noriko Goji,1
Chantal Paquet,2 Matthew C. Thomas,1 Timothy W. Janzen,1
Cesar I. Bin Kingombe,3 Arnold J. Kell,2 and Kristen R.
Hahn1
1 Lethbridge Laboratory, National Centres for Animal Disease,
Canadian Food Inspection Agency,P.O. Box 640, Township Road 9-1,
Lethbridge, AB, Canada T1J 3Z4
2 Emerging Technologies Division, National Research Council, 100
Sussex Drive, Ottawa, ON, Canada K1A 0R63 Sir F. G. Banting
Research Centre, Health Canada, 251 Sir Frederick Banting Dr.,
Tunney’s Pasture,Ottawa, ON, Canada K1A 0K9
Correspondence should be addressed to Kingsley K. Amoako,
[email protected]
Received 1 June 2012; Accepted 1 August 2012
Academic Editor: Dike O. Ukuku
Copyright © 2012 Crown. This is an open access article
distributed under the Creative Commons Attribution License,
whichpermits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Interest has recently been renewed in the possible use of Y.
pestis, the causative agent of plague, as a biological weapon by
terrorists.The vulnerability of food to intentional contamination
coupled with reports of humans having acquired plague through
eatinginfected animals that were not adequately cooked or handling
of meat from infected animals makes the possible use of Y. pestis
ina foodborne bioterrorism attack a reality. Rapid, efficient food
sample preparation and detection systems that will help overcomethe
problem associated with the complexity of the different matrices
and also remove any ambiguity in results will enable rapidinformed
decisions to be made regarding contamination of food with biothreat
agents. We have developed a rapid detectionassay that combines the
use of immunomagnetic separation and pyrosequencing in generating
results for the unambiguousidentification of Y. pestis from milk
(0.9 CFU/mL), bagged salad (1.6 CFU/g), and processed meat (10
CFU/g). The low detectionlimits demonstrated in this assay provide
a novel tool for the rapid detection and confirmation of Y. pestis
in food without the needfor enrichment. The combined use of the
iCropTheBug system and pyrosequencing for efficient capture and
detection of Y. pestisis novel and has potential applications in
food biodefence.
1. Introduction
Plague, caused by Yersinia pestis, has given rise to three
majorpandemics and is considered one of the most
devastatingdiseases in human history [1]. It still poses a
significant threatto human health and remains a current threat in
many partsof the world with about 2–3000 cases reported annually
[2].Due to the ease of transmission and the reappearance ofplague
in several countries, it has been recently categorizedas a
reemerging disease [3]. Furthermore, interest has beenrenewed in
the possible use of Y. pestis as a biologicalweapon by terrorists,
as it could cause mass casualties ifdispersed as an aerosol [4]. Y.
pestis is most commonlytransmitted through flea bites in animals
and the diseaseis manifested as bubonic, septicemic, or pneumonic
plague
[2, 5]. However, human plague has also been acquiredthrough
eating infected animals that were not adequatelycooked or through
the handling of meat from infectedanimals [6–13]. These reports
demonstrate that humanplague can be acquired through the
oropharyngeal route andhence poses a significant public health
risk. The vulnerabilityof food has been demonstrated by the
intentional contam-ination of salad bars in the United States with
Salmonellatyphimurium, and this makes the possible use of more
deadlyagents such as Y. pestis a possibility [14]. This concernis
exacerbated by the report of multidrug resistant strains[15] and
their potential use for bioterrorism in the humanpopulation. To
minimize this risk, the development of rapiddetection systems that
will enable the simultaneous detectionand confirmation of the
presence of Y. pestis is essential.
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2 Journal of Pathogens
Sample preparation and detection systems that will helpovercome
the problems associated with the complexity ofdifferent matrices
and also remove any ambiguity in resultswill enable rapid informed
decisions to be made regardingcontamination of food with biothreat
agents.
The recent development of next generation sequencingplatforms
has opened up new opportunities and helpedchange the direction of
microbial genomics and its appli-cation for pathogen detection
[16]. Sequencing-based tech-nologies are becoming rapid, cost
effective, and yield sub-stantially more genetic information which
helps to quicklymake informed decisions on foodborne disease
outbreaks.This was seen in the recent E. coli outbreak in
Europe,where the strain implicated was sequenced in a recordtime of
a few hours [17]. It also offers an added layer ofconfidence in the
identification of pathogens and provides anunambiguous detection
system for biodefence applicationssuch as foodborne bioterrorism
response. Pyrosequencingis a sequencing-by-synthesis method that
quantitativelymonitors the incorporation of nucleotides in real
time,through the emission of light following the
enzymaticconversion of pyrophosphate released during
nucleotideincorporation [18]. This technique generates similar
datato Sanger sequencing and is a rapid, reproducible,
high-throughput, user-friendly, and cost-effective method [19].
We have recently developed an immunomagnetic separa-tion (IMS)
assay for the efficient concentration of Bacillusanthracis spores
from different food matrices [20] and anovel sequence-based assay
using pyrosequencing for thespecific detection and antimicrobial
resistance gene profilingof Y. pestis [21]. Here, we present the
application of IMSand pyrosequencing based assays for the rapid,
specific, andsensitive detection and identification of Y. pestis
from foodmatrices such as milk, bagged salad, and processed
meat.This assay for Y. pestis detection is a significant
improvementover our previous work using the Pathatrix sample
prepa-ration system and real-time PCR [22] and demonstratesbetter
limits of detection without an enrichment step. Thecombination of
efficient immunomagnetic concentration ofbiothreat agents and
pyrosequence-based detection systemis novel and represents the
first report for detection andidentification of Y. pestis in food
with potential biodefenceapplication.
2. Materials and Methods
2.1. Bacterial Culture. Yersinia pestis KIM5- was culturedfrom a
glycerol stock on Tryptic Soy Agar plates (Difco,Becton Dickinson,
Sparks, MD, USA) supplemented with5% sheep blood (TSBAP) and grown
at 28◦C for 48 h. Asingle colony was subcultured in Brain Heart
Infusion (BHI)broth for 24 h at 37◦C. Cultures were serially
diluted in BHI,enumerated using TSBAP, and used for IMS in food
andpyrosequencing experiments.
2.2. Magnetic Bead Functionalization with Anti-Y.
pestisAntibodies. Two types of beads of different sizes and
surfacechemistries, consisting of the commercially available
Patha-trix beads (∼1 µm diameter, Life Technologies, Carlsbad,
CA, USA), and NRC-beads (300 nm diameter, NationalResearch
Council, Ottawa, ON, Canada), were used forfunctionalization. The
Pathatrix and NRC magnetic beadswere functionalized with anti-Y.
pestis antibody polyclonalrabbit anti-Y. pestis (Tetracore,
Rockville, MD, USA) or Y.pestis monoclonal Clone# M996145
(Fitzgerald IndustriesInternational, Acton, MA, USA) at a
concentration of1 mg/mL using the Pathatrix custom-coating kit with
slightmodifications [20]. The functionalized beads were adjustedto
a final concentration of 20 mg/mL and stored at 4◦C untiluse.
2.3. Comparison of IMS Methods, Antibodies, and Immuno-magnetic
Beads for the Capture of Y. pestis in Buffered PeptoneWater (BPW).
The two methods for capturing Y. pestis,Pathatrix Auto system (Life
Technologies, Carlsbad, CA,USA) and iCropTheBug systems (FiltaFlex
Ltd., Almonte,ON, Canada), were used as previously described [20].
Tocompare each machine for capture efficiency of Y. pestis, 1 mgof
Pathatrix immunomagnetic beads (IMBs) functionalizedwith rabbit
anti-Y. pestis and 50 mL of BPW (pH 7.2)containing ∼5 CFU/mL of Y.
pestis were mixed for 1 h,after which the beads were magnetically
captured from thesolution. The two different antibodies (monoclonal
andpolyclonal) were used to functionalize the Pathatrix beadsand
investigated for sensitivity with the iCropTheBug system.
The Pathatrix beads and NRC beads functionalized withthe Rabbit
anti-Y. pestis antibody were compared againstone another in the
same fashion as the antibody com-parison described above. One
milligram of functionalizedbeads was mixed with 50 mL of BPW
containing Y. pestis(∼2.5 CFU/mL) and captured.
The captured beads for all experiments were washed 3times in
washing buffer, resuspended in PBS buffer, plated onTryptic Soy
Blood Agar Plates (TSBAPs), and incubated for48 h at 28◦C for
colony enumeration. The experiments/assayswere run in triplicate
and plated in duplicate.
2.4. Data Analysis. Data for the IMS experiments wereanalysed by
dividing the total number of Y. pestis cellscaptured by the total
number of cells added to the BPW, andexpressed as percent recovery.
The total number of Y. pestiscells added was determined by plate
enumeration of preparedstock prior to each run. Standard deviations
were calculatedfrom the mean results of the replicate
experiments.
2.5. Preparation of Spiked Food Samples. Whole milk (3.25%milk
fat), processed meat (black forest ham), and prewashedbagged salad
(romaine lettuce) were purchased from a localgrocery store and used
for the food-spiking experimentsas previously described [20].
Briefly, Y. pestis cultures weregrown to a concentration of 107
CFU/mL and serially dilutedto 102–104 CFU/mL. Cells were added to
50 mL of wholemilk to achieve a cell inoculation of 0.1–7 CFU/mL of
Y.pestis. For bacterial capture in solid foods, 50 g of sliced
blackforest ham and 50 g of bagged salad were separately placedinto
a stomacher bag. The samples were inoculated with 0.3-1150 CFU/g of
Y. pestis cells and hand massaged to evenly
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Journal of Pathogens 3
distribute the bacteria throughout the food. Fifty millilitresof
BPW was added (1 : 1 dilution w/v) and the mixture wasstomached
with the Stomacher 400 Circulator (Seward Ltd.,West Sussex, UK).
The liquid was further passed through asponge filter and a 50 µm
stainless steel mesh filter using avacuum pump and the filtrate was
collected for analysis.
2.6. IMS of Y. pestis from Spiked Food Samples. Following
thepreparation of spiked food samples, 50 mL of the preparedfood
sample was mixed with 1 mg (50 µL) of Pathatrixbeads functionalized
with Rabbit anti-Y. pestis polyclonalantibody. The beads were mixed
and captured accordingto the iCropTheBug method as previously
described [20].Experiments involving each food matrix and bacterial
con-centration were done in duplicate.
2.7. DNA Preparations from Spiked Food Samples. The prepa-ration
of DNA from samples captured from the differentfoods was done as
previously described [22]. Briefly, 50 µLof bead samples captured
from food were lysed by vortexingvigorously and heating at 95◦C in
a thermal cycler. Followinga brief centrifugation, 3.5 µL of the
supernatant was usedas template for PCR amplification and then
followed bypyrosequencing analysis. The PCR primers and
reactionconditions are indicated in our previous work [21].
2.8. Pyrosequencing Analysis. Genomic DNA from Y. pestissamples
isolated from food were analysed using ourpreviously described
pyrosequencing assay [21]. Briefly,biotinylated PCR products were
bound to streptavidin-coated sepharose beads (GE Healthcare,
Piscataway, NJ)and the beads were then resuspended in annealing
buffercontaining 0.3 µM of the sequencing primer. Pyrosequencingwas
performed using the Pyro Gold Q24 reagents intriplicate, using
dispensations based on the target sequencewith the Pyromark Q24
system. Raw data files wereimported into Pyromark Q24 software
(version 2.0; QiagenInc.
[http://www.qiagen.com/products/pyromarkq24.aspx])for analysis
following pyrosequencing. Sequence data thatpassed the quality
check, as determined automatically bythe software, were compared to
the GenBank database(http://www.ncbi.nlm.nih.gov/genbank/) using
the sequencesearch function in Geneious (version 5.3.5; Biomatters
Inc.[http://www.geneious.com/]) to verify identity.
3. Results
3.1. Immunomagnetic Capture of Y. pestis Cells in BPW.The
polyclonal antibodies showed a better recovery ofY. pestis, with
efficiencies of 46–56%, when comparedto the monoclonal antibody
with 40–48% (Figure 1(a)).The iCropTheBug system showed a better
recovery of Y.pestis, when compared to the Pathatrix Auto system
whichhad efficiencies of 26–38% (Figure 1(a)). A comparisonof the
two beads indicated that the Pathatrix beads weremore efficient in
the recovery of Y. pestis bacterial cellsthan the NRC beads (Figure
1(b)). The Pathatrix beadsfunctionalized with polyclonal Rabbit
anti-Y. pestis antibody,
0
20
40
60
80
100
Rec
over
y (%
)
iCropTheBug and polyclonal
iCropTheBugand monoclonal
Pathatrix Auto and polyclonal
Concentration method and antibody
(a)
0
20
40
60
80
100
Rec
over
y (%
)
Pathatrix NRC
Immunomagnetic bead
(b)
Figure 1: Comparison of different recovery methods,
antibodiesand immunomagnetic beads for the recovery of Y. pestis.
(a) Patha-trix beads functionalized with polyclonal and monoclonal
antibod-ies were compared for Y. pestis recovery using the
iCropTheBug.The iCropTheBug and Pathatrix Auto systems were
comparedfor recovery using Pathatrix beads functionalized with
polyclonalantibody. (b) Pathatrix beads and NRC beads
functionalized withpolyclonal antibodies were compared using the
iCropTheBug.
in combination with the iCropTheBug method, showed themost
sensitive IMB/antibody combination (Figure 1).
3.2. Pyrosequencing of Y. pestis Samples Captured from Food.The
pyrosequencing of Y. pestis cells captured from thethree different
food matrices, conducted for targets Ypc4,Ypcaf1M1, and Yppst1,
yielded reads identical to thoseobserved in our previous report
[21] (Figure 2). As previ-ously observed, they yielded BLAST
results exclusive to Y.pestis and thus confirmed the identification
of Y. pestis.
3.3. Limit of Detection in Food Samples. The limits of
detec-tion for the three food matrices were determined
usingpyrosequencing (Table 1). These results indicate
detectionlimits of 0.9 CFU/mL, 1.6 CFU/g, and 10 CFU/g for
milk,bagged salad, and processed meat, respectively.
4. Discussion
The advent of novel-sequencing technologies, such as
pyro-sequencing, provides tools for the generation of sequence
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4 Journal of Pathogens
Consensus
Identity
Consensus
Identity
(1) Yppst1
Yppst1/Y. pestis Kim5-
(3) Milk (0.9 CFU/mL)
(1) Ypcaf1M-1
Ypcaf1M-1/Y. pestis Kim5-
(2) BPW (0.4 CFU/mL)
Consensus
Identity
(1) Ypc4
Ypc4/Y. pestis Kim5-1 10 20 30 40 43
1 10 20 30 40 43
1 10 20 30 40 43
71756267
71665943
58586572
Read length(bp)
Read length(bp)
Read length(bp)
(2) BPW (0.4 CFU/mL)
(2) BPW (0.4 CFU/mL)
(5) Ham (10 CFU/g)
(4) Salad (1.6 CFU/g)
(5) Ham (10 CFU/g)
(4) Salad (1.6 CFU/g)
(3) Milk (0.9 CFU/mL)
(5) Ham (10 CFU/g)
(4) Salad (1.6 CFU/g)
(3) Milk (0.9 CFU/mL)
Figure 2: Pyrosequence alignments from Y. pestis isolated from
food samples. Pyrosequencing reads for Y. pestis targets including
Ypc4(chromosome), Yppst1 (pPCP1 plasmid), and Ypcaf1M-1 (pMT1
plasmid) are shown with limit of detection from milk, ham, and
saladsamples.
information which helps in the detection of pathogens andenables
their rapid confirmation/identification. The use ofthese novel
tools is further enhanced by the availability ofwhole genome
sequences that provide unprecedented geneticinformation for the
generation of specific molecular markersfor diagnostic
applications. These markers, if carefullyselected could be used to
discriminate between closely relatedpathogenic strains and could be
used in the specific detectionand identification of microbial
contamination in food. Thedetection of Y. pestis in food matrices
using real-time PCRhas been previously reported [22], however, the
limitsof detection reported required further improvement.
Thepotential for contamination with very low bacterial numbersin
food matrices necessitates the development of methodsfor efficient
capture from food matrices. The present workexplored the use of a
novel IMS as an efficient capturemethod and pyrosequencing for the
detection and confirma-tion of Y. pestis directly from food without
enrichment.
The use of IMS for the efficient capture of pathogensfrom food
has received wide attention [22–26]. There islimited information on
capture and detection of Y. pestisin food [22] and, therefore, a
need to explore this further.In a recent publication [20], we
described the use of theiCropTheBug system as a novel immunocapture
method forthe concentration of anthrax spores from food. Using
thissame capture method, we investigated the use of different
Table 1: Detection limits of Y. pestis KIM5- in
experimentallyinoculated food matrices using IMS and
Pyrosequencing.
Sample matrix Ypc4 Yppst1 Ypcaf1M1
BPW (CFU/mL) 0.4 0.4 0.4
Milk (CFU/mL) 0.9 0.9 0.9
Salad (CFU/g) 1.6 1.6 1.6
Ham (CFU/g) 10 10 10
magnetic beads and antibodies for the capture of Y. pestiscells.
Here, we show that the highest capture efficiency isassociated with
the use of the Pathatrix beads in combinationwith a polyclonal
antibody (Figure 1(a)). This high captureefficiency is reflected in
the limits of detection observed.The effects of bead size and
different antibodies on captureefficiency have been previously
discussed [20, 27–30]. Resultsfrom the study suggest that even
though beads with small sizepresent a large surface area to volume
ratio, capture efficiencymay be reduced due to the small magnetic
core. Hence,the Pathatrix beads (which are much bigger) may
possessa larger magnetic core than the NRC beads (Figure 1(b))and
thus reflect a higher capture efficiency associated withthis bead
type. Thus, there is a trade-off for bead size;too small is
detrimental to magnetic effect, while too large
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Journal of Pathogens 5
appears to have some decreased capture efficiency. In
ourprevious study on B. anthracis, we showed that
polyclonalantibodies demonstrate a much higher efficiency in
thecapture of anthrax spores than monoclonal antibodies
[20],however, the results obtained in the current study for
Y.pestis suggest that the recoveries associated with the
twoantibodies are comparable (Figure 1(a)). The differences maybe
due to the antigenic targets used for the generation ofthe
antibodies. Information to substantiate this is lacking,as they are
both commercial and proprietary issues do notallow the disclosure
of the antigenic targets used. Furtherstudies using Y. pestis
strains possessing different mutationsin surface markers such as
the F1 antigen are required todelineate the capture specificity of
the antibodies.
Food is vulnerable to intentional contamination andthe tainting
of salad bars in the USA with Salmonellatyphimurium highlights this
risk [14]. There are very fewsample preparation methods that do not
rely on enrichmentprior to detection. The IMB mixing and recovery
systemalso play a key role in IMS. Two methods for mixing
andrecovery of the Y. pestis cells with IMB were compared.
ThePathatrix Auto system is currently one of the commonlyused
methods for the magnetic concentration of pathogensfrom food
matrices [22, 31], however, it had a relativelylow recovery when
compared to the iCropTheBug system(Figure 1). This is similar to
what has been seen in otherstudies [20, 32].
Pyrosequencing has been used for the detection andtyping of
several microbes [33–35]. The pyrosequencingreads observed in the
present study show consistently highsequence identities to the
expected sequences, and thereforereinforce the reliability of the
assays as a confirmatorytool. Typical runs were completed in about
60 minutesand hence offer a rapid sequence based detection
methodwith unprecedented limits of detection for Y. pestis in
afoodborne application (Table 1) [22]. In this study, all
liquidmatrices showed detection limits of 0.4–0.9 CFU/mL Y.
pestiscells, while the solid matrices ranged between 1.6–10
CFU/g(Table 1). Previous work done on Y. pestis in milk and
groundbeef showed detection levels of 101 CFU/mL in milk and102
CFU/g in ground beef without enrichment [22]. Previousreports
indicate the limit of detection is 103 CFU/mL for theIMS and
detection of Bacillus stearothermophilus spores fromfood and
environmental samples [36] while Shields et al.showed recovery of
B. anthracis spores as low as 1 CFU/mLfrom food without enrichment.
The low detection limitof the assay demonstrated in the present
study representsa significant improvement over those derived from
ourprevious work using real-time PCR [22] and provides a noveltool
for the rapid detection and confirmation of Y. pestis infood
without the need for enrichment.
This study has further demonstrated that pyrosequenc-ing is a
proven technology for sequence-based identificationand the
technology has an unprecedented set of propertiesthat makes it
uniquely suited to, and a highly powerfultool for, biodefence
applications. The technology is lessexpensive, time consuming, and
labor intensive, as wellas easier to perform than conventional
Sanger sequencing[19, 37]. To our knowledge, this is the first
report on the
use of pyrosequencing for the direct detection from foodsamples.
The combined use of the iCropTheBug system withpyrosequencing is
novel for Y. pestis capture and detection infood and offers a new
tool with an added layer of confidencefor biodefence
applications.
Acknowledgments
The authors thank Fanliang Kong for his technical
assistance.They also thank Dr. Joseph Hinnebusch (NIH,
Hamilton,Montana, USA), for kindly providing the Y. pestis
KIM5-strain. The authors acknowledge the useful suggestions ofDrs.
Oliver Lung and Soren Alexandersen regarding themanuscript. This
work was funded by the Defence ResearchDevelopment Canada Centre
for Security Science, Chemical,Biological, Radiological, Nuclear
and Explosive ResearchTechnology Initiative (CRTI) Grant CRTI
08-0203RD.
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